1. Field of the Invention
The invention relates to a method of ascertaining the mutual binding of a first reactant and a second reactant, which method comprises the following steps:
providing a substance comprising the first reactant in a holder so that the substance is adsorbed at the inner side of the holder, PA1 adding a solution containing the second reactant to the holder, and PA1 irradiating the holder with electromagnetic radiation and detecting whether a parameter of radiation from the holder has changed with respect to said parameter of the radiation incident on the holder. PA1 a radiation source for supplying a radiation beam having at least one discrete frequency, PA1 a support for a holder to accommodate a first and a second reactant, PA1 a detection system for detecting radiation from the holder, which device is characterized in that the detection system is adapted to detect only radiation having a frequency which differs essentially from said at least one discrete frequency.
The invention also relates to a device for performing this method. In known methods, the radiation parameter which may change is, for example, the color or the state of polarization.
2. Description of the Related Art
The method is often used as an antibody virus detection in medical diagnostics for detecting infectious diseases at an early stage, with the virus being the first reactant and the antibody being the second reactant. An antibody, or possibly several antibodies in succession, are added to a substance containing an unknown virus so as to detect this virus. Moreover, for example, for developing medicines, a material which may be a possible antibody for the virus may be added to a known virus so as to ascertain whether this antibody reacts with the virus. The biological substance which is examined by means of the method may be human or animal tissue, blood or secretion.
Due to the considerable risks which may be involved in viral infections, which may occur, for example during pregnancy or birth, virus-antibody tests are nowadays performed in large numbers.
Therefore, there is a need for inexpensive and rapid virus detection methods, all the more because after a test with a first antibody and after it has been found that the virus does not react to this antibody, a second test and possibly subsequent tests, each time with a different antibody, must be performed.
For detecting the presence of a virus, the method described in the opening paragraph is generally used in which, after addition to the substance of the solution with antibody to which Rhodamine 6G molecules are bound, an incubation time is observed in which the virus can react with the antibody.
Subsequently, the holder with the substance and the antibody is washed several times, for example four times, so that all antibodies which have not reacted with the substance are removed. Subsequently, it is ascertained by means of irradiation with light, whether there are still fluorescent Rhodamine 6G molecules present, hence whether there is still antibody. This antibody is then bound to the substance, in other words, it has reacted with a virus. If no fluorescence is observed, the substance does not contain a virus matching the antibody.
A great drawback of this method is that the washing operation must be performed frequently, which is time consuming and involves a waste of antibodies so that the method cannot be performed in situ. Moreover, the detection method is not very reliable.
To be able to detect in situ and to avoid the washing steps, alternative methods have been proposed which are based on optical techniques such as the detection of the quantity of light reflected by the substance, or of the change of the state of polarization of the light coming from the substance (ellipsometry) or of a technique known as the Fourier-transformed infrared (FTIR) technique.
PCT Patent Application No. 94/03774 describes a number of these techniques for detecting, inter alia, viruses which to this end are contacted with a thin layer in which antibodies are present and, if they react with an antibody, cause a change of an optical property such as the reflection coefficient, the polarization selectivity or the color selectivity.
For a reliable virus detection, only radiation from the surface of the antibody which is in contact with the substance to be examined should be detected. However, in the above-mentioned in situ detection methods, radiation from the holder may reach the detector, which radiation does not originate from this surface, hence of Rhodamine 6G molecules which are bound to antibody which has not reacted with the virus. Since no distinction can be made between Rhodamine 6G molecules which are bound to an antibody which has reacted with a virus and molecules which are bound to antibody but has not reacted with a virus, said in situ methods are not very reliable either.